The present invention relates to new antisecretory factors having fluid transport and/or inflammatory reactions regulating properties as well as polynucleic regulating properties, and polynucleic acids coding therefor, and the use thereof.
All cells and tissues of the body are critically dependent on a constant and normal fluid environment in combination with an adequate blood supply. Derangement of one or both of these supporting systems may rapidly become fatal. Concerning fluid imbalance, two principally different systems exist:
A. edema, which is characterised by the abnormal accumulation of fluid in the intercellular tissue spaces or body cavities, or
B. dehydration, which, in a strict sense, means loss of water only, but is in fact commonly used to describe the combined loss of water and ions.
The most common forms of either edema or dehydration are:
diarrheas, inflammatory bowel diseases, brain edema, asthma, rhinitis, conjunctivitis, arthritis, glaucoma, various forms of pathological intracranial pressure (increase or decrease), pressure alteration in the middle ear such as Morbus Meniere, dermatitis, chemical or physical derangement of the skin and skin adjacent glands such as mastitis, various forms of endocrine disorders, such as diabetes insipidus. Conn""s syndrome, Cushing""s syndrome and Morbus Addison, kidney diseases such as pyelonephritis and glomerulonephritis, metabolic diseases such as myxedema and acute intermittent porphyria, side effects during treatment with various drugs such as anti-diabetics, tricyclic antidepressants, cytostatics, barbiturates, narcotics and narcotic analogues.
Diarrhea is caused by a change in the permeability in the gut for electrolytes and water. This disturbance is often caused by bacterial enterotoxins such as those produced by Escerichia coli, Campylobacter jejuni, Vibrio cholerae, Shigella dysenteriae and Clostridium difficile. The disturbance could also be caused by intestinal inflammation. Since the uptake of water is coupled to the uptake of electrolytes and nutrients, animals with frequent diarrhea suffers from malnutrition, resulting in retardation of the daily weight gain in the growing animal. The body counteracts these reactions by neuro-hormonal mechanisms such as the release of somatostatin and opiate peptides from interneurons in the intestinal mucosa. These polypeptides are capable of reversing fluid secretion and diarrhea.
The recently described antisecretory factor (AF) has been partially purified from pig pituitary gland and shown to reverse pathological secretion induced by various enterotoxins. High levels of AF in sow milk protect the suckling piglets against neonatal diarrhea.
Antimicrobial drugs have been widely used in the treatment of diarrhea in both human and veterinarian medicine. They are also used as feed additives for pigs, calves and chicken. However, due to the rapid development of resistant bacteria in the gut, the use of antibiotics against enteritis is generally not accepted in human medicine and their use is also diminishing in veterinarian medicine.
Other antidiarrheal drugs counteract the secretion in the intestinal mucosa. Since these drugs are directed against the host animal, it is unlikely that resistance against the drugs will develop. These types of drugs include nerve-active drugs like phenothiazines and thioxanthenes. Due to some serious side effects these types of drugs have not been accepted for treatment of diarrhea in most countries. Other drugs are derivatives of opiates like codeine and loperamide. Since these drugs mainly acts by inhibiting intestinal mobility, they also inhibit the clearance of pathogenic bacteria from the gut and should definitely not be recommended against dysenteric bacteria or parasites. Derivatives of somatostatin have been introduced recently, but have so far a limited use due to difficulties in the administration of the drugs and possible interactions with the endocrine regulation of growth.
The antisecretory factor (AF) has so far not been used directly for treatment of diarrhea or malnutrition due to the difficulties involved in obtaining a pure preparation of this protein. However, it has been possible to induce similar proteins in domestic animals which have been given a specific feed (SE Patent No. 9000028-2). Pigs given this feed obtained high levels of AF-like proteins and had a significant increase in the daily growth rate compared to matched controls. AF in rats challenged with toxin A from C. difficile protects not only against intestinal secretion but also against inflammation and bleeding in the gut.
A major object of the present invention is to provide a new recombinant protein and homologues and fragments (peptides) thereof for use in normalizing pathological fluid transport. These proteins and peptides are collectively called antisecretory factors (AF). The use of AF also partly inhibits, or totally eliminates the development of inflammatory reactions of various aetiologies. Reconstitution back to normal (fluid transport or inflammation) is obtained by the use of proteins or peptides. Further the AF proteins or peptides are effectively absorbed via various mucus membranes without losing in potency (when compared to intravenous administration). Consequently, a multitude of treatment regimens exist, and a correctly administrated protein or peptide make it possible to rapidly reconstitute a deranged fluid (water and ion) balance, an inflammatory reaction, or both.
In summary, the recombinant AF (rAF) and the homologues and fragments thereof could be used for immunodetection, as feed additive for growing animals and as antidiarrheal and drugs against diseases involving edema, dehydration and/or inflammation.
The objects of the present invention are the following:
A recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof.
A fragment of the recombinant protein shown in SEQ ID No. 2, which fragment is chosen from the group comprising
a) amino acids nos. 35-42
b) amino acids nos. 35-46
c) amino acids nos. 36-51
d) amino acids nos. 36-80
e) amino acids nos. 1-80
of the amino acid sequence shown in SEQ ID No. 2.
A peptide X1VCX2X3KX4R corresponding to the fragment comprising the amino acids no. 35-42 of the recombinant protein shown in SEQ ID No. 2, wherein X is I or none, X2 is H, R or K, X3 is S, L or another neutral amino acid and X4 is T or A.
Antibodies against a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof.
A protein binding to antibodies specific to a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof.
A composition for normalising pathological fluid transport and/or inflammatory reactions comprising as an active principal an effective amount of the recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof.
Use of a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof for manufacturing a composition for normalising pathological fluid transport and/or inflammatory reactions.
Feed for normalising pathological fluid transport and/or inflammatory reactions in vertebrates, comprising as an active agent a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof, or an organism capable of producing such a protein or homologues or fragments thereof.
A process of normalising pathological fluid transport and/or inflammatory reactions in vertebrates, comprising administering to the vertebrate an effective amount of a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof, or an organism producing said protein or homologues or fragments.
Use of specific antibodies against a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof, for detecting said protein or fragments in organisms.
Nucleic acids coding for a recombinant protein having essentially the sequence shown in SEQ ID NO. 1, or homologues or fragments thereof.
Use of nucleic acids coding for a recombinant protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof, for producing corresponding proteins or homologues or fragments.
Use of probes or primers derived from nucleic acids coding for a recombinant protein having essentially the sequence shown in SEQ. ID No. 2, or homologues or fragments thereof, for detecting the presence of nucleic acids in organisms.
Vector comprising nucleic acids coding for a recombinant protein having essentially the amino acid sequence shown in SEQ. ID NO. 2, or homologues or fragments thereof.
Host except human comprising a vector including nucleic acids coding for a recombinant protein having essentially the amino acid sequence shown in SEQ. ID No. 1, or homologues or fragments thereof.
A strain of an organism except human capable of producing a protein having essentially the amino acid sequence shown in SEQ ID No. 2, or homologues or fragments thereof.
As organisms capable of producing the recombinant protein use can be made of different types of organisms, such as recombinant bacteria and eucaryotic organisms, such as yeast, plants and vertebrates except humans.
Despite ten years of attempts to purify AF by conventional biochemical techniques, it has not been possible to obtain AF in a homogeneous form. However, by means of a new procedure of preparing a semipure AF for immunisation and selecting antiserum by means of an immunohistochemical method a suitable antiserum was chosen. With this antiserum it has now been possible to clone recombinant human cDNA expressing AF in E. coli. 
The sequence of the new cDNA was determined and shown to be unique. By knowledge of this sequence, oligonucleotide probes were constructed which hybridise with human and porcine pituitary RNA. The size of this RNA, about 1400 basepairs, complies with the size of the sequenced cDNA comprising 1309 basepairs plus a poly(A)tail. A partial cDNA sequence from rat pituitary gland has been shown to be identical with that of the human cDNA reflecting a ubiquitous structure conserved in AF genes from different species. This resemblance makes it possible to use the same oligonucleotide probes to identify AF-coding RNA and DNA from different species.
It has furthermore been possible to express the rAF in a biological active form. The AF protein in form of a fusion protein with glutathione S-transferase was expressed in large amounts in E. coli and purified to homogeneity by affinity chromatography. After cleavage of the fusion protein with thrombin, the recombinant AF (rAF) was shown to be extremely potent, 44 ng (10xe2x88x9212 mol) giving a half-maximal inhibition of cholera toxin-induced fluid secretion in rat intestine.
By gene technique smaller fragments of rAF was produced. The activity was shown to reside in a small sequence consisting of 7 to 8 amino acids. This was confirmed by help of chemical solid phase synthesis by which technique an octapeptide was produced and shown to be almost as biological potent as rAF on molar basis. With help of site directed synthesis a variety of sequences within the active site was constructed and replacements of certain amino acids shown to be possible without abolishing the biological activity.
The fluid secretion was measured by the intestinal loop model: a section (loop) of the small intestine is ligated by means of two satures; in the loop a certain amount of enterotoxin is injected. If antisecretory drugs are tested they are injected between one hour before and two hours after toxin challenge. The injection was made by three different routes; intravenously, intraintestinally and intranasally. The fluid is accumulating in the loop 5 h after toxin challenge. The secretion is calculated from the weight of the accumulated fluid per cm intestine.
The sequence of the protein was determined both directly by amino acid sequencing and indirectly by deduction from the cDNA sequence.
Recombinant AF seems to exert very little toxic or systemic effects since no obvious toxic reactions were noted in rats given 100 fold higher doses than that causing half-maximal inhibition. Since it is efficient when injected in the small intestine it could be administrated perorally.
The recombinant AF inhibits secretion also when injected after toxin challenge in contrast to the preparations of natural AF tested which seem to efficient only when injected before the toxin. Thus, rAF could be used both prophylactically and therapeutically.
Further, rAF and its peptide fragments were shown to inhibit cytotoxic reactions and inflammation in the gut caused by toxin A from Clostridium difficile. By help of a dye permeability test rAF and its fragements were shown to reverse pathological permeability changes induced by cholera toxin not only in the intestinal mucusa but also in plexus choroideus which regulates the fluid pressure in the brain.
Antisera against rAF were produced in rabbits and used in enzyme-linked immuno assays (ELISA). This assay might be used to measure AF in body fluids or feed.
A method of purifying antibodies against AF (natural or recombinant) by means of affinity chromatography on columns with agarose coupled rAF is reported below.
The antibodies were also shown to be efficient for detection of AF in tissue sections by means of immunohistochemical techniques and for detection of AF in Western-blot.